Selection and study of a Candida molischiana mutant derepressed for β-glucosidase production

Abstract A mutant strain of Candida molischiana was selected. Analysis of the exocellular activity of Candida molischiana 35M5N grown on different carbon sources revealed that the biosynthesis of β-glucosidase is derepressed in this yeast strain. The strain is not a hyper-producer mutant. There were no observed differences in the endocellular and parietal activities of the wild and mutant strains. However, the mutant strain produced 35-fold more enzyme than the wild-type in the culture medium with glucose as carbon source. When glucose was used as carbon source, the mutant strain produced 90% more exocellular enzyme than when cellobiose was used as the carbon source.     

Production of β-glucosidase using immobilised<Emphasis Type="Italic"> Piromyces</Emphasis> sp. KSX1 and<Emphasis Type="Italic"> Orpinomyces</Emphasis> sp. 478P1 in repeat-batch culture

Two anaerobic fungi, one a monocentric strain (Piromyces sp. KSX1) and the other a polycentric strain (Orpinomyces sp. 478P1), were immobilised in calcium alginate beads and cultured in sequential batches where spent medium (containing 0.25% cellobiose) was repeatedly drained and replaced. β-Glucosidase production with KSX1 was maintained for 45 days over six repeated batch cultures yielding a maximum level of 107 mIU/ml. For 478P1, β-glucosidase production was maintained for 30 days over four repeated batches yielding a maximum level of 34 mIU/ml. Although repeat-batch cultures of KSX1 produced more β-glucosidase than strain 478P1, the maximum specific β-glucosidase produced from these immobilised cultures was similar. The immobilised polycentric strain proved to be operationally superior to strain KSX1, as strain 478P1 did not produce any growth in the culture liquor. Electronic Publication     

Exocellular β-glucosidase inducibility in a mutant strain ofCandida wickerhamii derepressed for endocellular β-glucosidase production

Summary Candida wickerhamii produces one endocellular and one exocellular -glucosidase. Both enzymes are repressed by glucose in the wild-type strain. In the M7 mutant ofC. wickerhamii, which was previously demonstrated to be derepressed for endocellular -glucosidase biosynthesis, the exocellular -glucosidase is derepressed and hyperproduced when cellodextrins are added to the culture medium. This enzyme, which was produced constitutively in the wildtype, has thus become inducible in the M7 mutant strain. The interest of this strain for industrial production of -glucosidases is discussed.

---

Resumen Candida wikerhamii produce dos -glucosidasas: una endocelular y otra exocelular. Ambos enzimas son reprimidos por glucosa en la cepa salvaje. Al añadir celodextrina al medio de cultivo del mutante M7 deC. wickerhamii, en el cual se ha demostrado ya la desrepresión de la síntesis de -glucosidasa endocelular, se desreprime la -glucosidasa extracelular obteniéndose una hiperproducción de este enzima. Dicho enzima que era producido de forma constitutiva en el tipo salvaje, se ha convertido en inducible en la cepa mutante M7. Se discute el interés de esta cepa para la producción industrial de -glucosidasas.

---

Résumé Candida wickerhamii produit deux -glucosidases, l'une endo- et l'autre exocellulaire. Les deux enzymes de la souche sauvage sont réprimées par le glucose. Le mutant M7, chez qui il a été antérieurement constaté que la synthèse de la -glucosidase endocellulaire est déréprimée, l'enzyme exocellulaire est elle aussi déréprimée et hyper-produite lorsque des cellodextrines sont ajoutées au milieu de culture. Cette enzyme, qui est constitutive chez la souche sauvage, est donc devenue inductible chez le mutant M7. Cette souche est intéressante pour la production industrielle de -glucosidases.

     

Type I β-lactamases ofEnterobacter cloacae and resistance to β-lactam antibiotics

The interaction of type-I β-lactamases fromEnterobacter cloacae with diverse β-lactam compounds was examined. The ability of penicillin and cefoxitin to induce β-lactamase production in this strain was assessed. The effect of β-lactamase inhibitors was measured on β-lactamase extracts and on intact cells.E. cloacae 78 strain is a stably derepressed mutant showing limited susceptibility to a number of antibiotics except imipenem. Imipenem would therefore be the appropiate choice for therapy of infections caused by stably derepressed mutants ofEnterobacter sp. producing type-I β-lactamases.     

Citric acid production from cellobiose by 2-deoxyglucose-resistant mutant strains of Aspergillus niger in semi-solid culture

Citric acid production from cellobiose by Aspergillus niger was studied by a semi-solid culture method using bagasse as a carrier. From the parental strain Yang no. 2, mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glucose as a carbon source were induced. The representative mutant strain M155 was selected and subjected to further mutation. The new series of mutant strains showing resistance to DG on minimal medium containing cellobiose as a carbon source was induced, and among them the best mutant strain C192 showed higher citric acid productivity than Yang no. 2 in semi-solid culture when glucose was used as a carbon source. Moreover, in semi-solid culture, the strain C192 produced 49.6 g/l of citric acid, 1.6 times as much citric acid as Yang no. 2 produced, from 100 g cellobiose/l and showed enhanced -glucosidase production. In shake culture, the extracellular -glucosidase activity of C192 was higher than that of Yang no. 2 when not only cellobiose but also glucose and glycerol, catabolite repressors, were used as a carbon source. These results indicate that mutant strains such as C192 are insensitive to catabolite repression. Correspondence to: S. Usami     

Aerobic and anaerobic alcohol dehydrogenases in Escherichia coli

Abstract Mutants unable to use ethanol for carbon and energy were counterselected from an ethanolutilizing mutant of Escherichia coli K12 derepressed for alcohol dehydrogenase (ADH). Mutants of one class were devoid of ADH activity under anaerobic conditions but exhibited aerobic activities comparable to those of wild-type E. coli. Mutants of a second class exhibited ADH activity levels intermediate between those of the wild-type and derepressed parent. Immunological studies showed that mutants of the former class synthesized far less ADH protein than did the derepressed parent while mutants of the latter class synthesized about the same amount. The ADH mutations in both classes were located within the previously described adh region which contains the structural gene for the activity that is derepressed in the parent. An Eth⁻ adh-lac fusion mutant with an insertion in the structural gene was also isolated and characterized. It exhibited no ADH activity under anaerobic conditions and wild-type levels under aerobic conditions. These data are consistent with the existence in E. coli of distinct aerobic and anaerobic ADH enzymes and a derepression of the anaerobic but not the aerobic enzyme in the ethanol utilizing strain.     

The inactivation of hexokinase activity does not prevent glucose repression in Candida utilis

Abstract High hexokinase activity was not related to glucose repression in Candida utilis IGC 3092. The addition of Cibacron Blue 3G-A to growing cells in batch culture led to a permanent in vivo hexokinase inactivation, decreased growth rate and inhibited alcohol dehydrogenase. Hexokinase inactivation up to 90% did not alleviate glucose repression of α-glucosidase, as has been described for Saccharomyces cerevisiae and other yeasts. Moreover, when cells were physiologically derepressed by growing them in a chemostat at low glucose concentrations, the highest hexokinase activity was shown by the derepressed cells, and decreased as repression increased. Thus, in our strain of C. utilis , hexokinase activity was inversely proportional to glucose repression.     

Characterization of β-glucosidase activity in yeasts of oenological origin

I. ROSI, M. VINELLA AND P. DOMIZIO. 1994. Three hundred and seventeen strains representing 20 species of yeasts were screened for the presence of β-glucosidase activity. All of the strains of the species Debaryomyces castellii, Deb. hansenii, Deb. polymorphus, Kloeckera apiculata and Hansenula anomala showed β-glucosidase activity, but only one of 153 strains of Saccharomyces cerevisiae. The other species behaved differently, depending upon the strain. The strains that hydrolysed arbutin were checked to localize the β-glucosidase activity. A strain of Deb. hansenii exhibited the highest exocellular activity and some wall-bound and intracellular activity. The β-glucosidase synthesis from this yeast was enhanced by aerobic conditions of growth, was repressed by high glucose concentration (9%) and occurred during exponential growth. The optimum conditions for enzymatic preparations of Deb. hansenii were between pH 4.0 and 5.0 and 40C. A high concentration of ethanol and glucose did not reduce the ezymatic activity. The enzymatic preparations of Deb. hansenii released monoterpenols and other alcohols from a grape glycoside extract.     

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of β-glucosidase activity in Escherichia coli

Abstract Genomic DNA fragments encoding β-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli . Two clones containing a common fragment encoded a polypeptide of 58000 Da. Cloned β-glucosidase, expressed in E. coli , showed activity against natural β-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids ( M _r 53896) which showed significant homology with β-glucosidases from glycosyl hydrolase family 1.     

Xylanases of anaerobic fungus <Emphasis Type="Italic">Anaeromyces mucronatus</Emphasis>

The anaerobic fungus Anaeromyces mucronatus KF8 grown in batch culture on M10 medium with rumen fluid and microcrystalline cellulose as carbon source produced a broad range of enzymes requisite for degradation of plant structural and storage saccharides including cellulase, endoglucanase, xylanase, α-xylosidase, β-xylosidase, α-glucosidase, β-glucosidase, β-galactosidase, mannosidase, cellobiohydrolase, amylase, laminarinase, pectinase and pectate lyase. These enzymes were detected in both the intra- and extracellular fractions, but production into the medium was prevalent with the exception of intracellular β-xylosidase, chitinases, N-acetylglucosaminidase, and lipase. Xylanase activity was predominant among the polysaccharide hydrolases. Extracellular production of xylanase was stimulated by the presence of cellobiose and oat spelt xylan. Zymogram of xylanases of strain KF8 grown on different carbon sources revealed several isoforms of xylanases with approximate molar masses ranging from 26 to 130 kDa.     

Double Mutants of Cellulomonas biazotea for Production of Cellulases and Hemicellulases following Growth on Straw of a Perennial Grass

Summary Deoxyglucose-resistant mutants of Cellulomonas biazotea secreted elevated levels of cellulases and xylanases. The production of β-glucosidase in the constitutive mutant was increased 5-fold over its parent strain. This mutant showed an approximately 1.6-fold enhanced productivity of extracellular endo-glucanase following growth on Leptochloa fusca over the mutant parent. Extracellular production of xylanase, filter-paper cellulase (FPase) and endo-glucanase (CMCase) were also altered in the mutant. Maximum volumetric productivities for xylanase, β-xylosidase, FPase, β-glucosidase and endo-glucosidase were 451, 98, 80, 95, and 143 IU l⁻¹ h⁻¹ which were significantly more than their respective values from the parental strains. The enzyme preparation of the mutants exhibited improved saccharification of kallar grass straw.     

Hydrolytic enzyme(s) production in Micrococcus roseus growing on different cellulosic substrates

Micrococcus roseus (G12) isolated from higher termite Odontotermes obesus gut exhibited cellulose digesting properties. A lignocellulosic substrate, rice husk induced endoglucanase, β-glucosidase, β-xylanase and β-xylosidase production. Besides rice husk, CMC also induced endoglucanase production. β-Glucosidase activity was quite pronounced when rice husk was supplemented with CMC or cellobiose. Both β-xylanase and β-xylosidase activities could be induced by xylan as well as xylobiose, whereas CMC induced partial activity. Endoglucanase and β-xylanase enzymes were secreted into the culture medium, whereas β-glucosidase and β-xylosidase activities were intracellular in nature. Enzyme production was subject to end product inhibition. The extracellular enzyme(s) possessed the potential to saccharify rice husk, xylan and CMC to reducing sugars.     

Influence of L-sorbose on the location of β-glucosidase in Trichoderma pseudokoningii

Abstract The effect of l -sorbose on growth, morphology, cell wall composition and β-glucosidase location has been examined with Trichoderma pseudokoningii . Sorbose-grown cultures exhibited a longer lag phase, a tendency to more frequent hyphal branching and showed a decreased cell wall content of β-1,3-glucan. In sorbose-containing cultures, a significant higher portion of total β-glucosidase was present in the culture fluid, whereas in sorbose-lacking control cultures the major part of activity was associated with the cell walls. The results support the previous hypothesis (Kubicek, C.P. (1982) Arch. Microbiol. 132, 349–354) that β-1.3-glucan is involved in cell wall binding of β-glucosidase in Trichoderma pseudokoningii .     

Interaction of virulent and non-virulent Rhodococcus equi human isolates with phagocytes, fibroblast- and epithelial-derived cells

Abstract Rhodococcus equi is a facultative, intracellular, Gram-positive coccobacillus, increasingly reported in pneumonia of AIDS-infected patients. We investigated killing resistance properties of human R. equi virulent and avirulent human strains. Avirulent β-lactam-susceptible strains had lower intracellular colony forming units after 45 min incubation in murine macrophages J774 and human monocyte-macrophage TPH-1 than those of virulent strains. Only virulent β-lactam-resistant strains persisted within macrophages for at least 18 min only. A β-lactam-resistant mutant was obtained from a β-lactam-susceptible strain after selection in a penicillin G-containing culture medium. This mutant strain, like the natural virulent strains, persisted within macrophages, harboured cell-associated appendages, produced phage-like particles and induced, after its intravenous inoculation, a chronic infection in BALB/c nude mice. Supernatant culture of virulent strains transferred partial macrophage-killing resistance properties to avirulent strains. The same supernatant was toxic for L-929, HeLa and Vero cell cultures. These supernatant effects were heat-inactivated, trypsin-inactivated and did not seem to be linked to phage-like particle presence. These data argue that virulence, β-lactam-resistance, and macrophage-killing resistance are associated in human R. equi isolates. Moreover, only virulent strains produced uncharacterized toxic factors.     

γ-ray induced mutagenesis ofCellulomonas biazotea for improved production of cellulases

A rifampin-resistant mutant ofCellulomonas biazotea secreted elevated levels of cellulasesin vivo. The cellulase production in the mutant was not inhibited in the presence of 5% glucose, cellobiose or glycerol in the solid medium. The mutant exhibited approximately two- to three-fold enhanced product yields and productivity of cellular β-glucosidase over the wild parent in shake-flask culture studies when grown on either cellulosic or lignocellulosic substrates. Extracellular production of filter paper cellulase (FPase) and endo-glucanase (CMCase) were also significantly (p≤0.05) altered. During growth of the mutant on α-cellulose, the maximum volumetric productivities for CMCase, FPase and β-glucosidase were 52, 23.3, and 15.2 IUL⁻¹ h⁻¹,i.e 118, 121, and 229% their respective values for the parental strain. Some enzyme properties of the mutant cellulases were altered. Mutant-derived cellulases produced higher yields of glucose arising by degradation of bagasse, wheat straw, and α-cellulose (1.53-, 1.57-, and 1.75-fold, respectively).     

Isolation and properties of mutants of the fungus Penicillium pinophilum with enhanced cellulase and β-glucosidase production

A number of mutant strains overproducing cellulase, β-glucosidase and xylanase enzyme were isolated from the cellulolytic fungus Penicillium pinophilum 87160iii after mutagenesis by u.v. irradiation and/or chemical treatment. Selection was carried out using either an agar-plate or an enrichment technique. Cellulase (filter paper-hydrolysing activity) production by some of the mutants in shake flask cultures was approximately four-fold higher than the wild-type strain; improvements in β-glucosidase production were of the order of eight- to-ninefold. The morphology of the mycelium of the mutants was quite different from that of the wild type. The mutants, for example, produced mycelium which was highly branched and thicker in cross section. In several of the mutants synthesis of xylanase and β-glucosidase was completely derepressed in the presence of glycerol, which was a known repressor of the synthesis of these enzymes. Several of the mutants produced β-glucosidase enzyme which showed altered kinetics of hydrolysis in the presence of inhibitors.     

Localisation of β-glucosidase in Trichoderma reesei cell walls with immunoelectron microscopy

Abstract Using ferritin-conjugated antibodies as an electron microscopic marker, β-glucosidase was localized within the cell walls of the imperfect fungus Trichoderma reesei QM9414. With different states of cell wall degradation obtained with a cell wall-lysing culture filtrate of Micromonospora chalcea , β-glucosidase was mainly detected within the outer, fibrous exopolysaccharide layer and the outer face of the plasma membrane.     

Changes in Particulate Neuraminidase Activity During Normal and Staggerer Mutant Mouse Development

Abstract: The activity of particulate neuraminidase (sialidase, EC 3.2.1.18) in wild-type mice and the neurological mutant Staggerer was studied during development. Peak activity of this enzyme was observed at postnatal day 3 (P3) in three tissues of normal mice: cerebellum, cerebrum, and liver. In Staggerer, however, neuraminidase peak activity was observed at P27 in the cerebellum, whereas the activity was close to normal in Staggerer cerebrum and liver. Activities of other glycosidases in Staggerer (α-glucosidase (pH 3.7), α- glucosidase (pH 6.0), N -acetyl-β-hexosaminidase, β-glucosidase, and β-galactosidase) did not show significant variation compared with wild-type at P27 in any of the three tissues. This indicates that the late activity peak of particulate neuraminidase activity in the Staggerer cerebellum is neuraminidase-specific and not due to a general increase of lysosomal enzymes.     

The role of the cellulolytic high molecular mass (HMM) complex of the anaerobic fungus Piromyces sp. strain E2 in the hydrolysis of microcrystalline cellulose

The anaerobic fungus Piromyces sp. strain E2 produces extracellular cellulolytic enzymes present both in a high molecular mass (HMM) complex or as individual proteins. Although the HMM complex was present in the culture fluid during all growth stages, the highest amounts of complex were obtained when cultures were harvested at the end of fungal growth. The complex obtained after gel-filtration chromatography on Sephacryl S-300 HR was found to be the major factor in hydrolysis of cellulose to glucose (sole product, up to 250 mM). The complex was very stable as demonstrated by identical hydrolysis patterns with fresh preparations or preparations stored at 4° C for 2 months. From inhibition experiments with gluconic acid lactone and glucose, it was concluded that the HMM complex must contain at least one glucohydrolase. SDS-PAGE analysis revealed that a partially purified HMM complex was composed of at least ten polypeptides and contained numerous endoglucanases and one β-glucosidase. Received: 10 October 1996 / Accepted: 11 December 1996     

Purification and characterization of ββ-glucosidase from Aspergillus niger strain 322

An extracellular β-glucosidase enzyme was purified from the fungus Aspergillus niger strain 322 . The molecular mass of the enzyme was estimated to be 64 kDa by SDS gel electrophoresis. Optimal pH and temperature for β-glucosidase were 5·5 and 50 °C, respectively. Purified enzyme was stable up to 50 °C and pH between 2·0 and 5·5. The K_m was 0·1 mmol l⁻¹ for cellobiose. Enzyme activity was inhibited by several divalent metal ions.